Differential Laser Doppler (DLD) is a technique well known to physicists interested in fluid flow and microturbulence. Microscopic particles in moving fluids can scatter coherent laser light and the Doppler frequency shift provides a very accurate measure of fluid velocity.
A primary objective of this invention is to provide an improved apparatus for tracking, monitoring and identifying microbiota swimming in a medium, which is preferably quiescent, and to provide a sensitive method for rapidly measuring very small changes in their motility and direction of movement. Other parameters such as the average size of the individuals, and the growth of the total number of organisms in suspension can also be monitored. The present invention can readily be applied to phytoplankton, zooplankton, bacteria, or miniature ecosystems containing a variety of suspended microscopic plants and animals. The invention is useful in studies in areas of ecology, medicine, cell biology, etc.
A number of articles of interest are in a text entitled "The Application of Laser Light Scattering to the Study of Biological Motion" edited by J. C. Ernshaw and M. W. Steer, copyright 1980, Plenum Publishing Corp. Several articles in this text deal with laser light measurements of motility of living cells and microorganisms, with particular reference being made to the article by J. C. Ernshaw entitled "Laser Doppler Velocimetry" which describes a differential laser doppler in which one of the beams was electronically down mixed to give effective frequency shifts as low as 10 kHz, and the article by J. P. Boon entitled "Motility of Living Cells and Microorganisms" which describes the effect of stimuli on the motility of cells.
The use of a frequency shifter in studies of reversing flows, high turbulance and fluctuating flows with zero mean velocity enables the determination of direction and magnitude of flow. Frequency shift also allows optimum matching of the doppler signal to the range of the frequency tracker.
Commercial frequency shifters, such as DISA frequency Shifter Model 55N10 allows up to 54 settings (.+-.9 mHz) to help optimize the signal. However, as indicated above, the lowest shift available is 10 kHz. It is an important objective of this invention to accurately identify and define doppler frequencies corresponding to the swimming velocities of microbiota and these are commonly found to be less than 20.times.10.sup.-6 msec.sup.-1 (see Table 1, W. Nultsch "Movements in Biochemistry and Physiology of Algae", 1974, pg. 865), corresponding to doppler frequencies substantially less than 1 kHz.
According to the present invention, the shift is related to the motility range of the microorganisms. Thus, the frequency shifter according to the present invention is at a value of about 5.5 kHz. This provides velocity resolution at magnitudes &lt;20.times.10.sup.-6 msec.sup.-1, ten times better than Ernshaw et al. (e.g. +0.3 msec.sup.-1). This enhanced resolution is critical for studies of microorganisms where subtle changes in velocities in the order of 1.times.10.sup.- 1 msec.sup.- can be caused by various applied stimuli. The earlier frequency shift to 5.5 kHz is not absolute, and shift ranges for specific biota are intended to be encompassed in this invention. Thus, an important feature of the invention is in the use of a multi-beam differential laser doppler system in which the frequency shift of one of the beams relative to the others is very low, in this preferred embodiment circa 5.5 kHz. Another feature of the invention is that specific frequency shifts are related to the motility characteristics of specific microbiota. In the most complete manifestation of this invention, apparatus to analyze frequencies below 10 kHz is included together with optional shifts appropriate to that very low range.
The invention has particular advantage and usefulness when used to describe the effects of exogenous stimuli. Electric fields, magnetic fields, and e.m. radiation can be applied to the sample volume without interrupting the analysis. Data already accumulated shows dramatic and previously unrecognized effects upon application of external fields to seawater suspensions of single celled marine algae and shellfish sperm.
The measurement format bears some resemblance to spectrophotometry; the same (static or flow) cell with transparent input and exit ports admits split laser beams which cross within the sample volume to form virtual fringes. Scattered light from microbiota crossing those fringes is picked up by a photomultiplier and processed electronically to provide an input to a computer data file. When interrogated this file yields histograms, x-y plots of individual algal velocities or group statistics, and spectra which report the various motions perceived by the laser light including translational and rotational modes. Velocities reported may span the range from 10 to less than 10.sup.-6 msec, allowing the characterization of the complete body kinetics of a culture of micro organisms. Unattended operation over many reproductive cycles has been demonstrated, and if provision is made to sustain a community of microbiota, it can be followed indefinitely.
Most importantly, coincidental stimuli can be studied at known points in the circadian cycle or some other temporal natural rhythm. The system can continuously track the diurnal or circadian rhythm. A 48 hour run would result in ca. 10.sup.6 data points defining the behavior of five to ten generations. One such run with the algae Tetraselmis is illustrated in FIG. 5. In the run each printed point was an average over a one (1) second interval. Each one second average was derived from circa 5 individual measurements. According to the invention, computer programs can impose fields and/or radiation at chosen intervals during the ongoing "baseline" run. Already data is available which dramatically illustrate the transient effect of a nearby spark discharge on Tetraselmis, and its subsequent rapid adaptation. Response appears to vary markedly with the phase of the circadian cycle. Similar observations have been made for the dinoflagellate Gyrodinium. Magnetic fields also cause dramatic changes in vector and velocity in some species. Coincident stimuli and the phase of the circadian cycle are likely to dominate biological response, just as they do in Homo sapiens. Thus, if an individual responds to a sound which they have never heard before, their nervous system response is likely to be quite different depending on the state of the other system variables. Is it a sunny noontime or a dark cold winter night? Quite different biological changes may result depending on these and other more subtle coincidental stimuli. Generally, the biological effects will be transient, but in extreme cases they may be permanent. The rapidity with which the invention's data collection proceeds makes wide band frequency scanning practical within a small increment of circadian phase angle.
The shift at 5.5 kHz does not improve the accuracy of the instrument per se. The 5.5 kHz shift does recognize that most of the swimming velocities (i.e. time for algae to get from A to B, as distinct from the substantial velocity of his flagellum, or its wobble), for minute organisms are found at the very low end of the 1-10 kHz range, and allows the signal to be electronically shifted towards the center of its range, which, according to this invention, allows laser doppler its best chance to perform. The accuracy of the present instrument still does not exceed plus or minus about 0.5% of the range maximum in the 8-bit mode, for individual measurements. The instrument may be further adapted to break the low range down into smaller pieces corresponding to the transmission of a larger number of bits of information. There is no technical obstacle here and in fact, this merely involves selecting the proper frequency counter. Utimately, the accuracy to which you can measure individual velocities is limited by the frequency counting mechanism (which can be so accurate that it is not a significant limitation) and by the trade-off between the number of bytes used to transmit the number and the corresponding transmission time. In prior art work, there was little interest in individual partical velocities. Investigators using LDV (laser doppler velocimeter) have been concerned with average velocities. The prior art was not concerned with the unique problem of tracking individuals accurately. Average velocity and overall direction on flow were the parameters from which they deduced their results. The larger the block used for averaging the less one is able to distinguish by/or multi-modality in a spectrum.
The data obtained using this invention is good to 10.3.times.10.sup.-6 msec.sup.-1. Because each point represents the average over the smallest integration interval available in the Disa instrument-one second. Depending on the algal concentration, circa 10 individual values will have been accumulated in the one second "stack" inside the integrator before the average is set out. Because averaging prior to transmission reduces the load on the transmission line Disa can afford to handle all this data in a 16-byte mode.
The limit improved by the accuracy of the voltage controlled (phase lock) oscillator which is the key element in the frequency monitoring circuit, sets up with a demonstrable accuracy of 10.3.times.10.sup.-6 msec.sup.-1 for one second averages. The instrument can be modified to improve the accuracy of frequency measurement in the 1-10 kHz range. For example, a Hewlett Packard frequency counter with 12-byte output would break the 1-10 kHz range into 2+2.sup.2 +2.sup.3 +2.sup.4 +2.sup.5 +2.sup.6 +2.sup.7 +2.sup.8 +2.sup.9 +2.sup.10 +2.sup.11 +2.sup.12 (e.g. about 4096 parts), which is all the accuracy one would ever ask for at the low velocities of interest.
A major aspect of the invention is the recognition that modified laser doppler permits very accurate measurement of individual motions (including flagella, whip, wobble, etc.) at very high data rates, allowing these data to be processed statistically to give higher significant measures of:
1) Temporal changes (such as rhythm, diur changes in direction and velocities).
2) The effects of exogenous stimuli including traces of foreign materials of importance in pollution ecology.
3) Identification of a species.
Possible modifications of reproductive behavior must be studied within an experimental framework which permits control of coincidental stimuli and circadian phase angle. Such effects could be beautifully explicit according to this invention using sample numbers in the 10.sup.-4 to 10.sup.-6 range.
In summary, the present invention can rapidly describe:
1. Frequency response,
2. Frequency/intensity relationships, and
3. The effect of coincidental stimuli and the variation of the effect with circadian phase angle.
These descriptions can be applied to:
1. Motility,
2. Individual growth rate,
3. Reproduction, and
4. Aging.
As noted earlier, the invention contemplates a wide range of exogenous stimuli and can be one or more stimuli, applied at a selected phase or time in the natural rhythm of the microbiota, selected from the following:
a) a magnetic field,
b) an electric field,
c) complex fields as from a spark discharge,
d) visible intensity/wavelength light in the visible spectrum,
e) x-rays,
f) ultraviolet light,
g) infrared light,
h) chemical,
i) pressure,
j) sonic,
k) radio frequency,
l) thermal.
It is well accepted that the motility of a plankton population is one good measure of its vigor and good health. The invention is valuable as a method of studying positive and negative exogenous effects on the mass of microbiota which occupy the most influential position at the base of the food web. Thus an important aspect of the invention is the use of differential laser Doppler in a biospectrometer, within which motility, reproduction and growth can be monitored at known circadian phase angles, in the presence of chosen exogenous stimuli such as electric and magnetic fields, trace chemical additions, or em radiation.
In the biospectrometric mode, far more than simple swimming velocity is detected using the invention. In fact, maximum sampling rate increases in proportion to velocity (because it takes less time to analyze enough virtual fringes to characterise the velocity to a given accuracy). The peaks on FIG. 7e for example, are identified by sampling rate 1 to show how easy it is to characterise the higher velocities/frequencies. It is believed that these high frequency motions will tend to be the most sensitive to exogenous stimuli.